This vignette outlines the steps of inference, analysis and visualization of cell-cell communication network for a single spatial transcriptomics dataset using CellChat. We showcase CellChat’s application to spatial transcriptomics data by applying it to a mouse brain 10X visium dataset (https://www.10xgenomics.com/resources/datasets/mouse-brain-serial-section-1-sagittal-anterior-1-standard-1-0-0). Biological annotations of spots (i.e., cell group information) are predicted using Seurat (https://satijalab.org/seurat/articles/spatial_vignette.html).

CellChat requires gene expression and spatial location data of spots/cells as the user input and models the probability of cell-cell communication by integrating gene expression with spatial distance as well as prior knowledge of the interactions between signaling ligands, receptors and their cofactors.

Upon infering the intercellular communication network, CellChat’s various functionality can be used for further data exploration, analysis, and visualization.

Load the required libraries

ptm = Sys.time()

library(CellChat)
library(patchwork)
options(stringsAsFactors = FALSE)

Part I: Data input & processing and initialization of CellChat object

When inferring spatially-proximal cell-cell communication from spatially resolved transcriptomic data, user also should provide spatial coordinates/locations of spot/cell centroids. In addition, to filter out cell-cell communication beyond the maximum diffusion range of molecules (e.g., ~250μm), CellChat needs to compute the cell centroid-to-centroid distance in the unit of micrometers. Therefore, for spatial technologies that only provide spatial coordinates in pixels, CellChat converts spatial coordinates from pixels to micrometers by requiring users to input the conversion factor.

CellChat requires four user inputs:

Load data

# Here we load a Seurat object of 10X Visium mouse cortex data and its associated cell meta data
load("/Users/suoqinjin/Library/CloudStorage/OneDrive-Personal/works/CellChat/tutorial/visium_mouse_cortex_annotated.RData")
# show the image and annotated spots
color.use <- scPalette(nlevels(visium.brain)); names(color.use) <- levels(visium.brain)
#> Loading required package: SeuratObject
#> Loading required package: sp
#> The legacy packages maptools, rgdal, and rgeos, underpinning the sp package,
#> which was just loaded, were retired in October 2023.
#> Please refer to R-spatial evolution reports for details, especially
#> https://r-spatial.org/r/2023/05/15/evolution4.html.
#> It may be desirable to make the sf package available;
#> package maintainers should consider adding sf to Suggests:.
#> 
#> Attaching package: 'SeuratObject'
#> The following object is masked from 'package:BiocGenerics':
#> 
#>     intersect
#> The following object is masked from 'package:base':
#> 
#>     intersect
#> Loading required package: Seurat
#> 
#> Attaching package: 'Seurat'
#> The following object is masked from 'package:igraph':
#> 
#>     components
Seurat::SpatialDimPlot(visium.brain, label = T, label.size = 3, cols = color.use)
#> Scale for fill is already present.
#> Adding another scale for fill, which will replace the existing scale.


# Prepare input data for CelChat analysis
data.input = Seurat::GetAssayData(visium.brain, slot = "data", assay = "SCT") # normalized data matrix
#> Warning: The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
#> ℹ Please use the `layer` argument instead.
#> This warning is displayed once every 8 hours.
#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
#> generated.

# define the meta data: 
# a column named `slices` should be provided for spatial transcriptomics analysis, which is useful for analyzing cell-cell communication by aggregating multiple slices/replicates. Of note, for comparison analysis across different conditions, users still need to create a CellChat object seperately for each condition.  
meta = data.frame(labels = Idents(visium.brain), slices = "slice1", row.names = names(Idents(visium.brain))) # manually create a dataframe consisting of the cell labels
meta$slices <- factor(meta$slices)
unique(meta$labels) # check the cell labels
#> [1] L2/3 IT Astro   L6b     L5 IT   L6 IT   L6 CT   L4      Oligo  
#> Levels: Astro L2/3 IT L4 L5 IT L6 IT L6 CT L6b Oligo
unique(meta$slices) # check the slice labels
#> [1] slice1
#> Levels: slice1

# load spatial transcriptomics information
# Spatial locations of spots from full (NOT high/low) resolution images are required. For 10X Visium, this information is in `tissue_positions.csv`. 
spatial.locs = Seurat::GetTissueCoordinates(visium.brain, scale = NULL, cols = c("imagerow", "imagecol")) 

# Scale factors of spatial coordinates
# For 10X Visium, the conversion factor of converting spatial coordinates from Pixels to Micrometers can be computed as the ratio of the theoretical spot size (i.e., 65um) over the number of pixels that span the diameter of a theoretical spot size in the full-resolution image (i.e., 'spot_diameter_fullres' in pixels in the 'scalefactors_json.json' file). 
# Of note, the 'spot_diameter_fullres' factor is different from the `spot` in Seurat object and thus users still need to get the value from the original json file. 
scalefactors = jsonlite::fromJSON(txt = file.path("/Users/suoqinjin/Library/CloudStorage/OneDrive-Personal/works/CellChat/tutorial/spatial_imaging_data_visium-brain", 'scalefactors_json.json'))
spot.size = 65 # the theoretical spot size (um) in 10X Visium
conversion.factor = spot.size/scalefactors$spot_diameter_fullres
scale.factors = data.frame(ratio = conversion.factor, tol = spot.size/2)

Create a CellChat object

USERS can create a new CellChat object from a data matrix or Seurat. If input is a Seurat object, the meta data in the object will be used by default and USER must provide group.by to define the cell groups. e.g, group.by = “ident” for the default cell identities in Seurat object.

NB: If USERS load previously calculated CellChat object (version < 2.1.0), please update the object via updateCellChat

cellchat <- createCellChat(object = data.input, meta = meta, group.by = "labels",
                           datatype = "spatial", coordinates = spatial.locs, scale.factors = scale.factors)
#> [1] "Create a CellChat object from a data matrix"
#> Create a CellChat object from spatial transcriptomics data... 
#> Set cell identities for the new CellChat object 
#> The cell groups used for CellChat analysis are  Astro L2/3 IT L4 L5 IT L6 IT L6 CT L6b Oligo
cellchat
#> An object of class CellChat created from a single dataset 
#>  648 genes.
#>  1073 cells. 
#> CellChat analysis of spatial data! The input spatial locations are 
#>                    x_cent y_cent
#> AAACAGAGCGACTCCT-1   3164   7950
#> AAACCGGGTAGGTACC-1   6517   3407
#> AAACCGTTCGTCCAGG-1   7715   4371
#> AAACTCGTGATATAAG-1   4242   9258
#> AAAGGGATGTAGCAAG-1   4362   5747
#> AAATAACCATACGGGA-1   3164   7537

Set the ligand-receptor interaction database

Our database CellChatDB is a manually curated database of literature-supported ligand-receptor interactions in both human and mouse. CellChatDB v2 contains ~3,300 validated molecular interactions, including ~40% of secrete autocrine/paracrine signaling interactions, ~17% of extracellular matrix (ECM)-receptor interactions, ~13% of cell-cell contact interactions and ~30% non-protein signaling. Compared to CellChatDB v1, CellChatDB v2 adds more than 1000 protein and non-protein interactions such as metabolic and synaptic signaling. It should be noted that for molecules that are not directly related to genes measured in scRNA-seq, CellChat v2 estimates the expression of ligands and receptors using those molecules’ key mediators or enzymes for potential communication mediated by non-proteins. Critically, synaptic signaling interactions can only be used when inferring neuron-neuron communication.

Users can update CellChatDB by adding their own curated ligand-receptor pairs.Please check our tutorial on how to do it.

CellChatDB <- CellChatDB.mouse # use CellChatDB.human if running on human data

# use a subset of CellChatDB for cell-cell communication analysis
CellChatDB.use <- subsetDB(CellChatDB, search = "Secreted Signaling", key = "annotation") # use Secreted Signaling

# use all CellChatDB except for "Non-protein Signaling" for cell-cell communication analysis
# CellChatDB.use <- subsetDB(CellChatDB, search = NULL, key = "annotation")

# use all CellChatDB for cell-cell communication analysis
# CellChatDB.use <- CellChatDB # simply use the default CellChatDB. We do not suggest to use it in this way because CellChatDB v2 includes synaptic signaling interactions can only be used when inferring neuron-neuron communication. 

# set the used database in the object
cellchat@DB <- CellChatDB.use

Preprocessing the expression data for cell-cell communication analysis

To infer the cell state-specific communications, we identify over-expressed ligands or receptors in one cell group and then identify over-expressed ligand-receptor interactions if either ligand or receptor is over-expressed.

We also provide a function to project gene expression data onto protein-protein interaction (PPI) network. Specifically, a diffusion process is used to smooth genes’ expression values based on their neighbors’ defined in a high-confidence experimentally validated protein-protein network. This function is useful when analyzing single-cell data with shallow sequencing depth because the projection reduces the dropout effects of signaling genes, in particular for possible zero expression of subunits of ligands/receptors. One might be concerned about the possible artifact introduced by this diffusion process, however, it will only introduce very weak communications. USERS can also skip this step and set raw.use = TRUE in the function computeCommunProb().

# subset the expression data of signaling genes for saving computation cost
cellchat <- subsetData(cellchat) # This step is necessary even if using the whole database
future::plan("multisession", workers = 4) 
cellchat <- identifyOverExpressedGenes(cellchat)
cellchat <- identifyOverExpressedInteractions(cellchat)
 
# project gene expression data onto PPI (Optional: when running it, USER should set `raw.use = FALSE` in the function `computeCommunProb()` in order to use the projected data)
# cellchat <- projectData(cellchat, PPI.mouse)
execution.time = Sys.time() - ptm
print(as.numeric(execution.time, units = "secs"))
#> [1] 14.51582

Part II: Inference of cell-cell communication network

CellChat infers the biologically significant cell-cell communication by assigning each interaction with a probability value and peforming a permutation test. CellChat models the probability of cell-cell communication by integrating gene expression with spatial locations as well as prior known knowledge of the interactions between signaling ligands, receptors and their cofactors using the law of mass action.

The number of inferred ligand-receptor pairs clearly depends on the method for calculating the average gene expression per cell group. Due to the low sensitivity of current spatial transcriptomics technologies, we suggest to use 10% truncated mean for calculating the average gene expression. The default ‘trimean’ method produces fewer interactions and will likely miss the signaling with low expression. In computeCommunProb, we provide an option for using different methods to calculate the average gene expression. Of note, ‘trimean’ approximates 25% truncated mean, implying that the average gene expression is zero if the percent of expressed cells in one group is less than 25%. To use 10% truncated mean, USER can set type = "truncatedMean" and trim = 0.1. The function computeAveExpr can help to check the average expression of signaling genes of interest, e.g, computeAveExpr(cellchat, features = c("CXCL12","CXCR4"), type = "truncatedMean", trim = 0.1).

Compute the communication probability and infer cellular communication network

To quickly examine the inference results, USER can set nboot = 20 in computeCommunProb. Then “pvalue < 0.05” means none of the permutation results are larger than the observed communication probability.

If well-known signaling pathways in the studied biological process are not predicted, USER can try truncatedMean with lower values of trim to change the method for calculating the average gene expression per cell group.

USERS may need to adjust the parameter scale.distance when working on data from other spatial transcriptomics technologies. Please check the documentation in detail via ?computeCommunProb.

ptm = Sys.time()

cellchat <- computeCommunProb(cellchat, type = "truncatedMean", trim = 0.1, 
                              distance.use = TRUE, interaction.range = 250, scale.distance = 0.01,
                              contact.knn = TRUE, contact.knn.k = 6)
#> truncatedMean is used for calculating the average gene expression per cell group. 
#> [1] ">>> Run CellChat on spatial transcriptomics data using distances as constraints of the computed communication probability <<< [2023-11-26 22:42:03.590081]"
#> The suggested minimum value of scaled distances is in [1,2], and the calculated value here is  1.30553 
#> [1] ">>> CellChat inference is done. Parameter values are stored in `object@options$parameter` <<< [2023-11-26 22:54:59.289621]"

# Filter out the cell-cell communication if there are only few number of cells in certain cell groups
cellchat <- filterCommunication(cellchat, min.cells = 10)

Infer the cell-cell communication at a signaling pathway level

CellChat computes the communication probability on signaling pathway level by summarizing the communication probabilities of all ligands-receptors interactions associated with each signaling pathway.

NB: The inferred intercellular communication network of each ligand-receptor pair and each signaling pathway is stored in the slot ‘net’ and ‘netP’, respectively.

cellchat <- computeCommunProbPathway(cellchat)

Calculate the aggregated cell-cell communication network

We can calculate the aggregated cell-cell communication network by counting the number of links or summarizing the communication probability. USER can also calculate the aggregated network among a subset of cell groups by setting sources.use and targets.use.

cellchat <- aggregateNet(cellchat)

execution.time = Sys.time() - ptm
print(as.numeric(execution.time, units = "secs"))
#> [1] 776.5397

We can also visualize the aggregated cell-cell communication network. For example, showing the number of interactions or the total interaction strength (weights) between any two cell groups using circle plot or heatmap plot.


ptm = Sys.time()

groupSize <- as.numeric(table(cellchat@idents))
par(mfrow = c(1,2), xpd=TRUE)
netVisual_circle(cellchat@net$count, vertex.weight = rowSums(cellchat@net$count), weight.scale = T, label.edge= F, title.name = "Number of interactions")
netVisual_circle(cellchat@net$weight, vertex.weight = rowSums(cellchat@net$weight), weight.scale = T, label.edge= F, title.name = "Interaction weights/strength")

netVisual_heatmap(cellchat, measure = "count", color.heatmap = "Blues")
#> Do heatmap based on a single object

netVisual_heatmap(cellchat, measure = "weight", color.heatmap = "Blues")
#> Do heatmap based on a single object

Part III: Visualization of cell-cell communication network

Upon infering the cell-cell communication network, CellChat provides various functionality for further data exploration, analysis, and visualization. Here we only showcase the circle plot and the new spatial plot.

Visualization of cell-cell communication at different levels: One can visualize the inferred communication network of signaling pathways using netVisual_aggregate, and visualize the inferred communication networks of individual L-R pairs associated with that signaling pathway using netVisual_individual.

Here we take input of one signaling pathway as an example. All the signaling pathways showing significant communications can be accessed by cellchat@netP$pathways.

pathways.show <- c("IGF") 
# Circle plot
par(mfrow=c(1,1))
netVisual_aggregate(cellchat, signaling = pathways.show, layout = "circle")

# Spatial plot
par(mfrow=c(1,1))
netVisual_aggregate(cellchat, signaling = pathways.show, layout = "spatial", edge.width.max = 2, vertex.size.max = 1, alpha.image = 0.2, vertex.label.cex = 3.5)


execution.time = Sys.time() - ptm
print(as.numeric(execution.time, units = "secs"))
#> [1] 1.618266

Compute and visualize the network centrality scores:

# Compute the network centrality scores
cellchat <- netAnalysis_computeCentrality(cellchat, slot.name = "netP") # the slot 'netP' means the inferred intercellular communication network of signaling pathways
# Visualize the computed centrality scores using heatmap, allowing ready identification of major signaling roles of cell groups
par(mfrow=c(1,1))
netAnalysis_signalingRole_network(cellchat, signaling = pathways.show, width = 8, height = 2.5, font.size = 10)


# USER can show this information on the spatial transcriptomics when visualizing a signaling network, e.g., bigger circle indicates larger incoming signaling
par(mfrow=c(1,1))
netVisual_aggregate(cellchat, signaling = pathways.show, layout = "spatial", edge.width.max = 2, alpha.image = 0.2, vertex.weight = "incoming", vertex.size.max = 4, vertex.label.cex = 3.5)

Visualize gene expression distribution on tissue

# Take an input of a few genes
spatialFeaturePlot(cellchat, features = c("Igf1","Igf1r"), point.size = 0.8, color.heatmap = "Reds", direction = 1)


# Take an input of a ligand-receptor pair
spatialFeaturePlot(cellchat, pairLR.use = "IGF1_IGF1R", point.size = 0.5, do.binary = FALSE, cutoff = 0.05, enriched.only = F, color.heatmap = "Reds", direction = 1)
#> Applying a cutoff of  0.05 to the values...


# Take an input of a ligand-receptor pair and show expression in binary
spatialFeaturePlot(cellchat, pairLR.use = "IGF1_IGF1R", point.size = 0.5, do.binary = TRUE, cutoff = 0.05, enriched.only = F, color.heatmap = "Reds", direction = 1)

NB: Upon infering the intercellular communication network from spatial transcriptomics data, CellChat’s various functionality can be used for further data exploration, analysis, and visualization. Please check other functionality in the basic tutorial named CellChat-vignette.html

Part V: Save the CellChat object

saveRDS(cellchat, file = "cellchat_visium_mouse_cortex.rds")
sessionInfo()
#> R version 4.3.1 (2023-06-16)
#> Platform: aarch64-apple-darwin20 (64-bit)
#> Running under: macOS Ventura 13.5
#> 
#> Matrix products: default
#> BLAS:   /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRblas.0.dylib 
#> LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0
#> 
#> locale:
#> [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
#> 
#> time zone: Asia/Shanghai
#> tzcode source: internal
#> 
#> attached base packages:
#> [1] stats     graphics  grDevices utils     datasets  methods   base     
#> 
#> other attached packages:
#>  [1] Seurat_5.0.0        SeuratObject_5.0.0  sp_2.1-0           
#>  [4] patchwork_1.1.3     CellChat_2.1.0      Biobase_2.60.0     
#>  [7] BiocGenerics_0.46.0 ggplot2_3.4.3       igraph_1.5.1       
#> [10] dplyr_1.1.3        
#> 
#> loaded via a namespace (and not attached):
#>   [1] RcppAnnoy_0.0.21       splines_4.3.1          later_1.3.1           
#>   [4] tibble_3.2.1           polyclip_1.10-4        ggnetwork_0.5.12      
#>   [7] fastDummies_1.7.3      lifecycle_1.0.3        rstatix_0.7.2         
#>  [10] doParallel_1.0.17      globals_0.16.2         lattice_0.21-8        
#>  [13] MASS_7.3-60            backports_1.4.1        magrittr_2.0.3        
#>  [16] plotly_4.10.2          sass_0.4.7             rmarkdown_2.24        
#>  [19] jquerylib_0.1.4        yaml_2.3.7             httpuv_1.6.11         
#>  [22] NMF_0.26               sctransform_0.4.1      spam_2.9-1            
#>  [25] spatstat.sparse_3.0-2  reticulate_1.31        cowplot_1.1.1         
#>  [28] pbapply_1.7-2          RColorBrewer_1.1-3     abind_1.4-5           
#>  [31] Rtsne_0.16             purrr_1.0.2            presto_1.0.0          
#>  [34] circlize_0.4.16        IRanges_2.34.1         S4Vectors_0.38.1      
#>  [37] ggrepel_0.9.3          irlba_2.3.5.1          listenv_0.9.0         
#>  [40] spatstat.utils_3.0-3   goftest_1.2-3          RSpectra_0.16-1       
#>  [43] spatstat.random_3.1-5  fitdistrplus_1.1-11    parallelly_1.36.0     
#>  [46] svglite_2.1.1          leiden_0.4.3           codetools_0.2-19      
#>  [49] tidyselect_1.2.0       shape_1.4.6            farver_2.1.1          
#>  [52] matrixStats_1.0.0      stats4_4.3.1           spatstat.explore_3.2-1
#>  [55] jsonlite_1.8.7         GetoptLong_1.0.5       BiocNeighbors_1.18.0  
#>  [58] ellipsis_0.3.2         progressr_0.14.0       ggridges_0.5.4        
#>  [61] ggalluvial_0.12.5      survival_3.5-7         iterators_1.0.14      
#>  [64] systemfonts_1.0.4      foreach_1.5.2          tools_4.3.1           
#>  [67] sna_2.7-1              ica_1.0-3              Rcpp_1.0.11           
#>  [70] glue_1.6.2             gridExtra_2.3          xfun_0.40             
#>  [73] withr_2.5.0            BiocManager_1.30.22    fastmap_1.1.1         
#>  [76] fansi_1.0.4            digest_0.6.33          R6_2.5.1              
#>  [79] mime_0.12              colorspace_2.1-0       scattermore_1.2       
#>  [82] tensor_1.5             spatstat.data_3.0-1    utf8_1.2.3            
#>  [85] tidyr_1.3.0            generics_0.1.3         data.table_1.14.9     
#>  [88] FNN_1.1.3.2            httr_1.4.7             htmlwidgets_1.6.2     
#>  [91] uwot_0.1.16            pkgconfig_2.0.3        gtable_0.3.4          
#>  [94] registry_0.5-1         ComplexHeatmap_2.15.4  lmtest_0.9-40         
#>  [97] htmltools_0.5.6        carData_3.0-5          dotCall64_1.0-2       
#> [100] clue_0.3-64            scales_1.2.1           tidyverse_2.0.0       
#> [103] png_0.1-8              knitr_1.43             rstudioapi_0.15.0     
#> [106] reshape2_1.4.4         rjson_0.2.21           nlme_3.1-163          
#> [109] coda_0.19-4            statnet.common_4.9.0   cachem_1.0.8          
#> [112] zoo_1.8-12             GlobalOptions_0.1.2    stringr_1.5.0         
#> [115] KernSmooth_2.23-22     parallel_4.3.1         miniUI_0.1.1.1        
#> [118] pillar_1.9.0           grid_4.3.1             vctrs_0.6.3           
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#> [124] car_3.1-2              xtable_1.8-4           cluster_2.1.4         
#> [127] evaluate_0.21          magick_2.8.1           cli_3.6.1             
#> [130] compiler_4.3.1         rlang_1.1.1            crayon_1.5.2          
#> [133] rngtools_1.5.2         future.apply_1.11.0    ggsignif_0.6.4        
#> [136] labeling_0.4.3         plyr_1.8.8             stringi_1.7.12        
#> [139] deldir_1.0-9           viridisLite_0.4.2      network_1.18.1        
#> [142] gridBase_0.4-7         BiocParallel_1.34.2    munsell_0.5.0         
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#> [148] RcppHNSW_0.5.0         future_1.33.0          shiny_1.7.5           
#> [151] highr_0.10             ROCR_1.0-11            broom_1.0.5           
#> [154] bslib_0.5.1